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Tamoxifen is widely used in the adjuvant treatment of estrogen receptor–positive (ER+) breast cancer and is an important drug for pre-menopausal women and post-menopausal patients who cannot tolerate aromatase inhibitors. Despite the clear clinical benefit in improving relapse-free and overall survival in these patients, an adverse effect of tamoxifen is a 2- to 7-fold increased risk of uterine cancer (UC) after 2-5 years of treatment. To date, the mechanism of tamoxifen-driven tumorigenesis is not well understood, and preventive approaches are lacking. Here, to molecularly characterize tamoxifen-associated uterine cancers (TA-UCs) and gain insights into their unique evolution, we performed whole-exome sequencing of 21 TA-UCs (discovery cohort) and droplet digital PCR (ddPCR) of an additional 40 TA-UCs (validation cohort) obtained from the ‘Tamoxifen Associated Malignancies: Aspects of Risk’ (TAMARISK) study. In addition, we used in vivo mouse models to: (i) further investigate tamoxifen-activated molecular pathways that may be involved in TA-UC tumorigenesis; and (ii) offer mechanistic insights. Overall, we discovered that TA-UCs were genomically similar to non–TA-UCs from The Cancer Genome Atlas (TCGA) project, with one profound exception: TA-UCs are characterized by a lower-than-expected frequency of mutations in two highly prevalent UC driver genes in the PI3K pathway: PIK3CA (14% [3/21] vs 48% [265/554] in non–TA-UC; P=0.003, Fisher’s exact test; Q=0.02, Benjamini-Hochberg FDR) and PIK3R1 (0%, [0/21] vs 31% [174/554]; P=0.001; Q=0.01). We used ddPCR in the independent TA-UC validation cohort and confirmed the low frequency of mutations in PIK3CA (7.5% [3/40] vs 21% [144/685] in control UCs from the Dana-Farber contribution to the AACR GENIE project; P=0.04). We next performed mouse in vivo studies and demonstrated that tamoxifen activated the PI3K pathway and increased cell proliferation in normal mouse uterine tissue through paracrine and autocrine effects, both of which were abrogated by the PI3K inhibitor alpelisib. Taken together, we describe a distinct and novel pathway of carcinogenesis in which tamoxifen acts as a driver event in the uterus and promotes tumor development in a mutation-independent manner. Indeed, tamoxifen may increase the risk of UC by activating the PI3K pathway, which can substitute for the early acquisition of oncogenic PIK3CA or PIK3R1 mutations observed in non–TA-UC tumors. Furthermore, the ability of a PI3K inhibitor to reduce cell proliferation in our mouse model raises the possibility that downregulating the PI3K pathway may prevent or significantly reduce TA-UC development, offering a potential future therapeutic and prevention strategy for specific high-risk patients undergoing tamoxifen therapy. Citation Format: Kirsten Kubler, Agostina Nardone, Shankara Anand, Daniel Gorvich, Marjolein Droog, Francisco Hermida-Prado, Tara Akshi, Avery S Feit, Gabriella Cohen, Gwen Dackus, Matthew Pun, Yanan Kuang, Justin Cha, Mendy Miller, William J Gibson, Cloud P Paweletz, Eliezer M Van Allen, Flora E van Leeuwen, Petra Nederlof, Harry Hollema, Quang-Dé Nguyen, Marian JE Mourits, Ignaty Leshchiner, Chip Stewart, Ursula A Matulonis, Wilbert Zwart, Yosef E Maruvka, Gad Getz, Rinath Jeselsohn. Tamoxifen instigates uterine cancer development by activating PI3K signaling and supersedes PIK3CA driver mutations [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS2-09.

Background: After decades of research on Ashkenazi Jewish (AJ) founder mutations in BRCA1 and BRCA2 (BRCA), there is still little known about genetics in non-AJ and Arab breast cancer populations in Israel and non-BRCA pathogenic variants (PV) in the total population. We used multigene panel testing (MGPT) to characterize the spectrum and prevalence of PV in cancer susceptibility genes in various Jewish and Arab demographic subgroups. Methods: A population-based cohort of women diagnosed with breast cancer before the age of 46 in a defined geographical area in Northern Israel was recruited as part of the BCINIS study and underwent MGPT. Probability matrices of proportion of PV by gene, and by ethnic sub-group (AJs, Sephardi Jews of North African origin, Sephardi Jews of other Eastern origins, Arabs), age-sub-group (35 and under, 36-45) and first-degree family history of breast, ovary or pancreatic cancer were calculated. Results: Overall, 1650 women were tested, including 1012 Jewish, 530 Arab and 108 non-Jewish/non-Arab women. We detected 375 PV among 363 women (22.7%): 212 in AJ (33.4% of AJs), 61 in Sephardi Jews (14.2% of Sephardi), and 69 in Arabs (13.0% of Arab). Only 162 of the PV (43.2%) were the BRCA AJ founders, another 39 BRCA PV (10.4%) were reported previously, while 15 BRCA PV (4.0%) are likely private. The remaining non-BRCA PV (42.4%) were distributed across 18 genes, with findings predominantly in CHEK2, MUTYH, ATM, BLM, ERCC3, PALB2, and FANCA. The prevalence of PV varied significantly across the ancestral subsets, from 95.5% probability in AJ women diagnosed before age 36 and with first degree family history to only 9.3% in Arab women diagnosed at age 36-45 and without family history. Conclusions: In this comprehensive, population-based, cohort of women with young onset breast cancer, the presence of PV in more than 15 genes is compelling and informs a necessarily broader approach to genetic testing and counseling. Citation Format: Gad Rennert, Bita Nehoray, Flavio Lejbkowicz, Sara Dishon, Shiri Kalet, Joseph Herzog, Thomas Slavin, Danielle Castillo, Kevin Tsang, Sharon Sand, Hedy S Rennert, Jeffrey Weitzel. A comprehensive germline genetic landscape in young Jewish & Arab women with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-07.

Background Antibody drug conjugates (ADC) are novel drugs linking potent payloads to antibodies targeting antigen-expressing tumors. Sacituzumab govitecan (SG), targeting Trop-2, is approved for metastatic triple negative breast cancer (TNBC); and trastuzumab deruxtecan, targeting HER2, is approved for HER2-positive and HER2-low metastatic breast cancer. To understand the potentially overlapping clinical landscape of Trop-2 and HER2 antigens, we evaluated RNA expression data in breast cancer from The Cancer Genome Atlas (TCGA) project. Methods TCGA dataset was assessed for Trop-2 and HER2 expression via processed RNA sequencing (RNA-seq) data of the corresponding genes TACSTD2 and ERBB2. Medium/high gene expression was assessed as >100 transcripts per million (TPM). Samples were classified HER2-low per ASCO/CAP guidelines. Gene expression across clinical parameters was assessed via one-way ANOVA. Results 1076 patients with primary breast cancer were included. The majority (59%) had both high TACSTD2 expression (TACSTD2hi) and high ERBB2 expression (ERBB2hi) (see Table). Median TACSTD2 expression was 572 TPM (IQR 349-666 TPM); median ERBB2 expression was 122 TPM (IQR 73-192 TPM). No significant difference was observed in TACSTD2 or ERBB2 expression among invasive ductal carcinoma, invasive lobular carcinoma, mixed histology, or other (p = 0.07, 0.23). No significant difference in TACSTD2 expression was noted between HER2-low and HER2-negative subtypes (p=0.34). Conclusions While SG is approved in TNBC, TACSTD2 is expressed across all breast cancer subtypes, including HER2-low, suggesting a broader population may benefit from Trop-2-targeted ADCs. Furthermore, given that over half of breast cancers have high expression of both TACSTD2 and ERBB2, additional studies are needed to understand the optimal sequencing of ADC-based therapies for patients with breast cancer. Table Patient subsets(HR = hormone receptor) n TACSTD2hi & ERBB2hi TACSTD2hi & ERBB2low TACSTD2low & ERBB2hi TACSTD2low & ERBB2low All 1,076 632 (59%) 394 (36%) 19 (2%) 31 (3%) Histology 843 Invasive ductal carcinoma 507 267 (32%) 215 (25%) 10 (1%) 15 (2%) Invasive lobular carcinoma 130 103 (12%) 24 (3%) 1 ( Citation Format: Arielle J. Medford, Nicholas J. Haradhvala, Neelima Vidula, Rachel Abelman, Laura M. Spring, Leif W. Ellisen, Gad Getz, Aditya Bardia. Overlapping expression landscape of antibody drug conjugate targets, trophoblast cell surface antigen 2 (Trop-2) & human epidermal growth factor receptor 2 (HER2), in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 960.

Women who harbor germline heterozygous mutations of BRCA1 or BRCA2 have a high risk of breast cancer. Our previous study showed that patient-derived, ostensibly normal BRCA2mut/+ luminal progenitor (LP) cells are more prone to exhibit sub-chromosomal copy number variations and associated DNA damage relative to non-carriers potentially reflecting early breast tumorigenesis. The clinically assessable biomarkers for early pathological changes of BRCA1/2 mutation in LP cells remain unknown. Single-cell RNA sequencing (scRNAseq) of LP cells of BRCA1/2 mutation carriers revealed enrichment of KIT expression (KIT+) and subsequent transcriptional factor activations were observed in LP cells of BRCA1/2 mutation carriers relative to non-carriers. Moreover, overrepresented pathway analysis uncovered that KIT+ BRCA-mutated LP cells were enriched in pathways involving DNA binding transcription activator activity and oxidative phosphorylation. These gene signature profiles were recapitulated in bulk RNA-seq of BRCA2 mutation carrier LP cells. Collectively, our preliminary data suggest such analyses may identify potential biomarkers of the risk prediction for early pathogenesis of BRCA1/2 mutation carriers. Following validation of our findings via tissue microassay analysis and clinical trials, we hope to eventually assist clinicians in their decision making of prophylactic surgeries for BRCA1/2 mutation carriers. Citation Format: Zuen Zen, Po-Han Lin, Siang Boon Koh, Kai Stewart, Nick Haradhvala, Aylin S Dedeoglu, Ilze Smidt, Akiko Suzuki, David Li, Win Thant, Sarah Mueller, Taisha Joseph, Veerle Bossuyt, Michael Lawrence, Gad Getz, Leif W. Ellisen. Genomics and transcriptomics profiling of BRCA mutation carriers revealed the landscape of early pathogenesis of BRCA1/2-associated breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 767.

Comprehensive characterization of tumor evolution is essential for understanding the drivers of metastasis and treatment resistance, which are still largely unknown. Studying evolution and resistance requires large cohorts of patients; for each patient, a comprehensive phylogenetic tree is used to follow clones over time and space. Deep whole-genome sequencing (WGS) enables the creation of more robust phylogenies, since (i) each clone is characterized by far more clone-specific mutations (compared to whole-exome or panel sequencing); and (ii) WGS yields more accurate clonal and sub-clonal allelic copy-number estimates, enabling more precise estimation of the fraction of cancer cells that harbor each mutation. Due to recent development of more affordable WGS by Ultima Genomics, we set out to test our ability to construct phylogenetic trees based on Ultima WGS. Ultima sequencing has demonstrated high sensitivity and specificity for detecting germline polymorphisms, but its performance on somatic mutation calling has not yet been rigorously studied. First, we adjusted our somatic mutation detection pipeline to work with Ultima-generated WGS data and tested its performance using well-studied cell line tumor/normal pairs. We assembled a ground truth set of somatic mutations based on deep sequencing data from multiple platforms for the breast cancer cell line pairs (HCC1954/HCC1954-BL and HCC1143/HCC1143-BL) and the melanoma tumor/normal cell line pair (COLO829/COLO829-BL). We found that mutation detection performance was on-par with other platforms for single nucleotide variations. Next, we identified 32 patients from various cancer types (breast, lung, cholangiocarcinoma, and melanoma) treated with a variety of therapies. We collected between 4 to 14 samples per patient at autopsy (total of 303), and sequenced them with Ultima to an average coverage of 63x (range 48x-91x). We identified mutations using our Ultima-adjusted WGS pipeline and constructed phylogenetic trees using our PhylogicNDT suite of tools. We then compared the results to trees previously constructed from mostly whole-exomes (and a few whole-genomes) sequenced on Illumina. We demonstrate that the trees generated from Ultima WGS data are consistent with the previously generated trees, and proved to be much more detailed. At commercialization, we anticipate that WGS for projects like this will be performed at ~1/6th the cost of current NGS offerings (on par with current WES costs). Therefore, it will enable larger studies and more accurate phylogenetic reconstruction, which will advance the study of tumor evolution and resistance. Citation Format: Julian Hess, Elizabeth Martin, Ilya Soifer, Hila Benjamin, Mendy Miller, Carrie Cibulskis, Brian P. Danysh, Matthew Coole, Stacey Gabriel, Dejan Juric, Doron Lipson, Gad Getz. Phylogenetic reconstruction across 303 metastatic tumor samples using Ultima whole-genome sequencing dramatically increases subclonal resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 213.

Lenvatinib is a clinically effective multi-kinase inhibitor approved for first-line therapy of advanced hepatocellular carcinoma (HCC). Although resistance against lenvatinib often emerges and limits its anti-tumor activity, the underlying molecular mechanisms involved in endogenous and acquired resistance are still unidentified. Endogenous (SNU449 and SNU475) and established acquired (Hep3B-LR and Huh7-LR) lenvatinib resistant cells, as well as knockout mice were used in this study. As a result, we recognized focal adhension kinase (FAK) as a critical contributor to lenvatinib resistance in HCC. The elevated expression of FAK was observed in both established and endogenous lenvatinib-resistant HCC cells. Furthermore, inhibition of FAK reversed lenvatinib resistance in vitro and in vivo. Mechanistically, FAK promoted lenvatinib resistance through regulating lysine deficient protein kinase 1 (WNK1) signaling pathway. WNK1 inhibitor WNK463 re-sensitized either established LR HCC cells (Hep3B-LR and Huh7-LR) or endogenous resistant HCC cells (SNU449 and SNU475) to lenvatinib treatment. In addition, overexpression of WNK1 desensitized parental HCC cells (Hep3B-P and Huh7-P) to lenvatinib treatment. Conclusively, our results establish a crucial role and novel mechanism of FAK in lenvatinib resistance, and suggest that targeting the FAK/WNK1 axis is a promising therapeutic strategy in HCC patients showing lenvatinib resistance. Citation Format: Wei Hou, Shaimaa A. Gad, Wei Qiu. Focal adhension kinase (FAK) confers lenvatinib resistance in hepatocellular carcinoma via regulation of lysine deficient protein kinase 1(WNK1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 389.

Background. Despite substantial progress in the treatment of HER2+ MBC, most patients (pts) still experience disease progression and cancer-related death. HER2-directed TKIs are highly effective therapies for pts with HER2+ MBC; however, an understanding of resistance mechanisms is needed. Pts receiving HER2-directed TKIs with cell-free DNA (cfDNA) sampling across the treatment spectrum present a unique opportunity to examine genomic alterations. Methods. Pts with biopsy-proven HER2+ MBC were selected from DF/HCC approved protocols for ultra-low pass whole genome sequencing if ≥1 cfDNA and/or tissue sample had been collected prior to and after at least six weeks of TKI treatment. Only pts with ≥ 1 sample with tumor fraction (TFx) ≥ 9.5% and ≥ 1 additional sample with TFx ≥ 4.5% were analyzed. Whole exome sequencing (WES) was performed on 19 tumor biopsies and 64 cfDNA samples from 25 pts. WES samples were analyzed, variants called and annotated, copy number profiles inferred, and TFx estimated. PhylogicNDT was used to detect the phylogenetic architecture, and clones were identified as growing, stable, shrinking, or truncal; mutational signatures were called using SignatureAnalyzer. After filtering likely benign variants, mutations were labeled likely resistance mechanisms if there was known evidence of resistance to anti-HER2 therapy in a human breast cancer cell line, breast cancer mouse models, or in vivo. Findings were correlated with pt clinicopathologic data. Results. In 4/8 pts with acquired resistance (TKI treatment ≥ 180 days), we identified alterations in previously characterized pathways and genes that could explain treatment resistance (e.g. PIK3CA, ERBB2, FGFR2). Mutations in growing subclones identified potential novel mechanisms of resistance and included inactivating mutations in CDK12, KMT2D, KMT2C, CHEK2, BRCA2, and FAT1 genes. In 7/17 pts with intrinsic resistance, we identified mutations in overlapping pathways and genes such as ERBB2 and PIK3CA, which were present in both growing subclonal and truncal clones. Four pts with hormone receptor positive disease - two of whom had not received prior aromatase inhibitors - had an activating ESR1 hotspot mutation (D538G). This raises the possibility that activating ESR1 mutations may be involved in resistance in these cases. Mutational signature analysis revealed a subset of samples with widespread APOBEC activation (with and without hyper mutation), subclonal HR/MMR-related signature, and capecitabine-related 5FU signature. Conclusions. Genomic analysis of paired samples from pts with HER2+ MBC identifies candidate resistance mechanisms to anti-HER2 TKIs and clonal evolution over time in the context of heterogeneity in treatment and sample timing. Additional studies will determine the functional role and clinical utility of assessing these alterations to overcome resistance. Citation Format: Heather A. Parsons, Conor Messer, Katheryn Santos, Brian P. Danysh, Melissa E. Hughes, Ashka Patel, Raquel A. Jacobs, Kara Slowik, Julian Hess, Chip Stewart, Kristy Schlueter-Kuck, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Nikhil Wagle, Jose Pablo Leone, Rachel Freedman, Laxmi Parida, Ian E. Krop, Gad Getz, Nancy U. Lin. Genomic mechanisms of resistance to tyrosine kinase inhibitors (TKIs) in HER2+ metastatic breast cancer (HER2+ MBC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3874.

Post-translational modifications (PTMs) play key roles in regulating cell signaling in health and disease processes. Recent advances in mass spectrometry enable comprehensive investigation of PTMs. However, identifying functionally relevant PTMs and protein domains implicated in disease remains a key challenge. We present CLUMPS-PTM, an algorithm for spatial clustering of PTMs to propose functional domains. We apply CLUMPS-PTM to the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset (one of the largest proteogenomic pan-cancer PTM datasets) comprising 1110 samples spanning 11 tumor types. First, we map over 14K phospho-sites and 13K acetyl-sites detected in the CPTAC dataset to protein data bank (PDB) files. We expand this to the predicted AlphaFold proteome and recover additional 12K phospho-sites and 10K acetyl-sites exceeding model confidence (pLDDT) of 70%. Consistent with previous research, phospho-sites are found on residues with lower AlphaFold prediction confidence than acetyl-sites due to their abundance in unstructured domains (p-value < 1e-4). To identify functionally relevant protein domains in the pan-cancer context, we characterize the 1110 samples by applying SignatureAnalyzer, a Bayesian non-negative matrix factorization method, to discover multi-omic patterns. This analysis identifies strong signatures associated with DNA damage response (DDR) and stratifies the CPTAC cohort into DDR-high (n=442) and DDR-low (n=668) groups. Next, we applied CLUMPS-PTM to differentially modified phospho/acetyl-sites between these groups. We identified 22 proteins (including GMPS, NCBP1, and ANPEP (CD13)) with significant clusters of differentially phosphorylated sites (q-value < 0.1) in DDR-high samples. ANPEP is a zinc-dependent aminopeptidase with a significant cluster (q-value = 0.0129) of differentially phosphorylated sites in the ERAP1-C domain that faces peptidase active sites known to have proliferative activity via the MAPK pathway. CLUMPS-PTM also identified a significant cluster of acetyl-sites on ARID1A (a SWI/SNF chromatin remodeler known to be mutated in cancer) in DDR-high samples (q-value = 0.085). The ARID1A cluster is on the C-terminal tail of the protein within the glucocorticoid receptor (GR) domain, an upstream regulator of proteins that increase catabolism, reduce inflammation, and increase cell survival. Increased acetylation may block a ubiquitination site at the C-terminus of the protein, resulting in increased protein levels, likely due to reduced ARID1A degradation, and increased GR signaling. Overall, CLUMPS-PTM is an open-source tool that allows near proteome-wide spatial analysis with the growing availability of PTM data. We anticipate that it will be useful to the broader proteomic community for the discovery of novel targets and generation of insights into functional mechanisms. Citation Format: Shankara K. Anand, Yifat Geffen, Yo Akiyama, Francois Aguet, Gad Getz. CLUMPS-PTM: Spatial clustering of post-translational modifications across cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3132.

Introduction Patients with hematological malignancies, including multiple myeloma (MM), experience sub-optimal responses to SARS-CoV-2 vaccination. Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM) are precursors to MM and exhibit altered immune cell composition and function. The SARS-CoV-2 pandemic and the subsequent population-wide vaccination represent an opportunity to study the real-life immune response to a common antigen. Here, we present updated results from the IMPACT study, a study we launched in November 2020 to characterize the effect of plasma cell premalignancy on response to SARS-CoV-2 vaccination (vx). Methods We performed: (i) ELISA for SARS-CoV-2-specific antibodies on 1,887 peripheral blood (PB) samples (237 healthy donors (HD), and 550 MGUS, 947 SMM, and 153 MM patients) drawn pre- and post-vx; (ii) single-cell RNA, T cell receptor (TCR), and B cell receptor (BCR) sequencing (10x Genomics) on 224 PB samples (26 HD, and 20 MGUS, 48 SMM, and 24 MM patients) drawn pre- and post-vx; (iii) plasma cytokine profiling (Olink) on 106 PB samples (32 HD, and 38 MGUS and 36 SMM patients) drawn pre- and post-vx; and (iv) bulk TCR sequencing (Adaptive Biotechnologies) on 8 PB samples from 4 patients (2 MGUS, 2 SMM) drawn pre- and post-vx. Results Patients with MGUS and SMM achieved comparable antibody titers to HD two months post-vx. However, patient titers waned significantly faster, and 4 months post-vx we observed significantly lower titers in both MGUS (Wilcoxon rank-sum, p=0.030) and SMM (p=0.010). These results indicate impaired humoral immune response in patients with MGUS and SMM.At baseline, the TCR repertoire was significantly less diverse in patients with SMM compared to HD (Wilcoxon rank-sum, p=0.039), while no significant difference was observed in the BCR repertoire (p=0.095). Interestingly, a significant increase in TCR repertoire diversity was observed post-vx in patients with SMM (paired t-test, p=0.014), indicating rare T cell clone recruitment in response to vaccination. In both HD and patients, recruited clones showed upregulation of genes associated with CD4+ naïve and memory T cells, suggesting preservation of the T cell response in SMM, which was confirmed by bulk TCR-sequencing in 4 patients.Lastly, by cytokine profiling, we observed a defect in IL-1β and IL-18 induction post-vx in patients with SMM compared to HD (Wilcoxon rank-sum, p=0.047 and p=0.015, respectively), two key monocyte-derived mediators of acute inflammation, suggesting an altered innate immune response as well. Conclusion Taken together, our findings highlight that despite the absence of clinical manifestations, plasma cell premalignancy is associated with defects in both innate and adaptive immune responses. Therefore, patients with plasma cell premalignancy may require adjusted vaccination strategies for optimal immunization. Citation Format: Yoshinobu Konishi, Romanos Sklavenitis-Pistofidis, Michelle P. Aranha, Ting Wu, Hong Yue, Elizabeth D. Lightbody, Mahshid Rahmat, Michael Timonian, Shohreh Varmeh, Daniel Heilpern-Mallory, Michael P. Agius, Nang K. Su, Jacqueline Perry, Erica Horowitz, Maya I. Davis, Anna V. Justis, Radosław P. Nowak, Mark Hamilton, Daniel Auclair, Catherine R. Marinac, Eric S. Fischer, Gad Getz, Irene M. Ghobrial. Altered immune response to vaccination in patients with plasma cell premalignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 796.

DNA-dependent protein kinase (DNA-PK) plays an important role in the repair of DNA double-strand breaks via the non-homologous end joining (NHEJ) pathway. In addition to its role in the NHEJ pathway, DNA-PK is also known to have an emerging role in cell cycle progression and mitosis. Some studies have shown that depletion or inhibition of DNA-PK inhibitors leads to a delay in the transition from metaphase to anaphase. The mitotic MTH1 inhibitor OXC-101(TH1579, karonudib) is a dual inhibitor that both targets MTH1 and disrupts tubulin polymerization in cancer cells, causing DNA damage and mitotic catastrophe in cancer cells. Based on the involvement of DNA-PK in mitosis, we hypothesized that depletion/inhibition of DNA-PK might enhance the DNA damage and mitotic catastrophic effect of OXC-101.To test our hypothesis, we performed an in vitro drug combination experiment using the DNA-PK inhibitor nedisertib with OXC-101 in non-small cell lung carcinoma NSCLC (H460,A549), uveal melanoma (MP -38, MP -41 and MP46), osteosarcoma (U2OS) and) and neuroblastoma (IMR32, Kelly, SKNFI) cancer cell lines and non-cancerous foreskin fibroblasts(VH-10, BjhTERT). We found cancer-specific synergistic drug interactions between nedisertib and OXC-101. Combined treatment of nedisertib and OXC-101 to H460 and MP46 increased expression of s10H3 (mitotic marker) and the apoptosis marker (cPARP) compared with single treatment. However, there was no additional increase in expression of the DNA damage markerγH2AX with combined treatment. We also performed annexin V staining by FACS on MP38 andMP48 cells and observed a significant increase in the apoptotic population with combination treatment compared with single treatment. Imaging live cells, we observed a significant delay or arrest in the mitotic phase followed by death of cancer cells in mitosis in response to combination treatment, whereas cells treated individually with nedisertib or OXC-101 continued to proliferate and progress to the next phase of the cell cycle. These data suggest that the DNA-PK inhibitor may enhance the cytotoxic effect of OXC-101specifically in cancer cells. In addition, we will conduct an in-depth mechanistic study of how depletion/inhibition of DNA-PK enhances the cytotoxicity of OXC-101 and validate the drug combination in an appropriate preclinical mouse model. Citation Format: Akhilesh Nagesh Danda, Helge Gad, Martin Scobie, Laura Martínez-López, Nuria Pastor Carrillo, Manuel Luis Orta, Thomas Helleday, Ulrika warpman berglund, Kumar Sanjiv. DNA-PK inhibition augment the cancer specific cytotoxicity of mitotic MTH1 inhibitor OXC-101 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6204.

Despite the introduction of a number of new therapeutic classes including targeted inhibitors, immunotherapy, and antibody-drug conjugates, the ability to achieve long-term disease control in the majority of patients with advanced cancer remains elusive due to the emergence of resistance. While our understanding of resistance mechanisms continues to expand through preclinical and clinical sequencing efforts, comprehensive approaches to map the distribution of potential resistance mechanisms in a given cell line and drug context require the development of novel approaches. Here, we describe the design of a platform for profiling cancer resistance at scale. Beginning with a single sensitive parental cancer cell line, the EGFR-mutant lung cancer line PC9, we optimize pre-existing diversity using a combination of single-cell subcloning and alkylator mutagenesis, and generate 50 independently evolved clones resistant to the EGFR inhibitor osimertinib. Profiling of this library via multiplexed whole genome sequencing (WGS) demonstrates convergent mutations in the MAPK pathway. Taking a similar approach, we profile the NTRK1-fusion-driven KM12 colon cancer cell line, yielding convergent, clinically observed, on-target resistance mutations to the NTRK inhibitor larotrectinib. Taken together, these results provide an initial proof-of-concept for the high-throughput generation of viable drug-resistant cancer clones. Future efforts to increase mutational diversity as well as assess additional drug and disease contexts will broaden the utility of this approach. Citation Format: Arvind Ravi, Sainikhil Sontha, Maisha Chowdhury, Jacob Smigiel, Matthew Rees, Brian Danysh, Jennifer Roth, Laxmi Parida, Eric Lander, Gad Getz. A platform for deep evolutionary profiling of cancer resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 143.

3D cancer cell cultures have enabled new opportunities for replacing compound testing in experimental animals and provide a basis for high content work-flows for drug testing. Here we present data obtained from 3D multicellular tumor spheroids (MCTS) composed of cancer cell lines, fibroblasts and macrophages. We cultured HT29 and SW480 CRC cell lines (as well as others) with fibroblasts and obtained heterospheroids that could be evaluated in cell viability and apoptosis assays, by flow cytometry and high content imaging (HCI). In addition, mature MCTS were embedded in paraffin and used for traditional biomarker characterization using immunohistochemistry and in situ hybridization. The cancer cell x fibroblast interactions promoted a tumor-like microenvironment that recruited monocytes isolated from primary donors. FACS analysis indicated that the infiltrating monocytes polarized into CD163-positive M2 macrophages in HT29 MCTS, and in pancreatic MIA PaCa-2 MCTS, using Pexidartinib and anti-CSF1R mAb, we found that monocyte infiltration was strongly CSF1R dependent. Preliminary data suggest that also infiltration of T cells into the heterospheroids is possible, thus making the models useful for the study of immune cell infiltration. Whole transcriptomic analysis of mono-cultures of SW480 cancer cells and fibroblast spheroids and co-cultures (MCTS) identified traditional cell-type-related transcripts as well as a group of de novo expressed genes induced by co-culturing, such as serpin peptidase inhibitor clade A1 (serpinA1) and inhibin-β A (INHBA). The NGS analysis as well as immunohistochemical staining also showed that SW480 expressed high levels of P-glycoprotein (P-Gp, MDR1) in contrast to the fibroblasts. On the other hand, the HT29 MCTS were negative for P-Gp. Histological analysis and the apoptosis assay showed that the fibroblasts were more sensitive to 5FU and cisplatin treatment than the cancer cells. We are currently testing if the P-Gp inhibitor, PSC-833, can re-sensitize the cancer cells to 5FU and cisplatin. In conclusion, we have developed 3D multicellular heterospheroids mimicking tumor-like microspheres that can be used for drug testing of traditional drug compounds, biologics and even immunotherapeutic agents. Citation Format: Boye Schnack Nielsen, Natasha H. Madsen, Jesper Larsen, Hjalte M. Larsen, Monika Gad, Kim Holmstrom. 3D cancer cell-fibroblast heterospheroids forming tumor-like microenvironments are valuable tools to study immune cell infiltration and as pre-clinical drug testing models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4566.

Background: Diffuse midline gliomas (DMGs) are a universally fatal brain tumor of childhood. While histone mutations are a critical tumor initiating event, they are insufficient to drive gliomagenesis. Histone mutations co-occur with somatic alterations in other pathways including TP53, MAPK, and MYC signaling. However, the mechanisms through which these pathways are activated have not been fully elucidated. Methods: We applied an integrative approach using transcriptomics, epigenetics, proteomics, in vitro cancer models, and in vivo mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. Results: We have recently found that a subset of DMGs aberrantly express FOXR2, a forkhead transcription factor. FOXR2 is both sufficient to enhance tumor formation, and necessary for FOXR2-expressing DMGs. While FOXR2 indeed enhances MYC protein stability, FOXR2 exerts oncogenesis through MYC-independent functions and specifically hijacks E26-transformation specific (ETS) transcriptional circuits and FOXR2 DNA-binding is highly enriched at ETS motifs. We have performed proteomic and phospho-proteomic analysis of FOXR2-expressing human neural stem cells to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells. Conclusion: Taken together, this study elucidates how FOXR2 interacts with ETS transcription factors to mediate oncogenesis, and further highlights a role for FOXR2 in activating ETS and MAPK signaling. Citation Format: Jessica W. Tsai, Paloma Cejas, Marissa Coppola, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Annarah Charles, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Volker Hovestadt, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. Dissecting mechanisms underlying FOXR2-mediated gliomagenesis in diffuse midline gliomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3562.

Osteosarcoma (OS) and Leiomyosarcoma (LMS) are sarcomas with complex genomes for which there has been limited progress in identifying new treatments and improving outcomes. Slow progress in OS and LMS is partially due to insufficient characterization of the genomic landscape. Generating large genomic datasets in OS and LMS is challenging because of the rarity of these sarcomas and recruitment barriers such as care fragmentation between institutions and specialties. The OS and LMS Project research studies aim to: 1) establish a network of engaged pediatric and adult participants with OS and LMS who will co-create a shared database of clinical, genomic, molecular, and patient reported data to enable research; 2) define the clinicogenomic landscape of OS and LMS; and 3) optimize the approach to direct patient engagement in cancer research. Count Me In, a research initiative with prior success in angiosarcoma, working with patients and advocates created websites (OSProject.org and LMSProject.org) where patients register and consent to participation. Within two months of launching, 233 patients age 6-79 from 149 Institutions have consented. Blood and saliva are collected from consented participants, tumor samples are obtained from pathology departments and medical records are requested from treating hospitals. WES and WGS of tumor and normal, and RNASeq of tumor is performed. ctDNA is obtained and sequenced. Results are shared with patient, advocacy, physician and research communities in several ways. Individual participants receive a shared learning report describing the somatic variants identified in their tumor from paired tumor-normal WES and are offered genetic counseling and clinical germline testing. Registered participants receive updates via email and Project websites. There are regular pre-publication data releases to the genomic data commons and to cBioPortal. A physician engagement committee meets regularly to discuss clinical insights and conundrums from shared learning reports and germline testing. Patient accrual over the next 3 years is anticipated to result in sequencing of 750 tumor-normal pairs and 500 ctDNA samples. Citation Format: Katherine A. Janeway, Suzanne George, Corrie Painter, Carrie Cibulskis, Taylor Cusher, Jordan Doucette, Elana Anastasio, Benjamin Zola, Ashley Mathews, Evelina Ceca, Maeve Smart, Beena Thomas, Jason Hornick, Alanna Church, Lorena Lazo De La Vega, Jill Stopfer, Sidney Benich, Ellen Sukharevsky, Sarah Winnicki, Brendan Reardon, Brian Crompton, Priscilla Merriam, Adrian Marino-Enriquez, Diane Diehl, Eliezer VanAllen, Judy Garber, Gad Getz, Stacey Gabriel, Timothy Rebbeck, Jennifer Mack, Nikhil Wagle. The Osteosarcoma and Leiomyosarcoma Count Me In Projects of the Cancer Moonshot funded PE-CGS Network directly engage patient participants in genomics research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6084.

Many cancers suffer from replication stress, oxidative stress, a dysfunctional redox status, and high levels of endogenous oxidative DNA damage. OXC-101 (karonudib, TH1579) targets these vulnerabilities by stopping the cancer cells in mitosis, generating ROS which increases oxidative stress and oxidative DNA damage, thereby killing the cancer cells. The aim of this study was to further investigate the underlying mechanism of OXC-101 and the cancer specific effects in solid cancers. Using e.g. microtubule polymerization assays, co-immunoprecipitation and immunofluorescence microscopy, we show a dual mechanism of action of OXC-101, disturbing microtubule function and inhibiting the nucleotide pool sanitizing enzyme MTH1. Furthermore, we demonstrate the importance of a functional spindle assemble checkpoint (SAC) for the response of OXC-101, since knocking down MAD2,using siRNA, or inhibiting SAC with reversine, results in treatment resistance. We show that the excess of oxidized nucleotides is cytotoxic when inhibiting or knocking out MTH1 in zebrafish embryos, and that pretreatment with ROS scavenger rescues the effect of OXC-101 in cancer cells. These data indicate that the increased incorporation of oxidized nucleotides into DNA is, together with the mitotic arrest, an important part of the mechanism of action and response. OXC-101 has a broad anti-cancer effect. In an ex vivo drug sensitivity study of 157 primary biopsies from solid cancer patients, OXC-101 was efficacious in a large proportion of the patient derived samples, with best responses in endometrial (73%), HNSCC (73%), bladder (70%) and prostate (83%) cancer samples. Biomarker searches for genetic alterations explaining responders/non-responders are presently on-going. In summary, the dual mechanism of action of the mitotic MTH1 inhibitor OXC-101 explains its strong anti-cancer effect. Citation Format: Ulrika Warpman Berglund, Helge Gad, Kumar Sanjiv, Ann-Sofie Jemth, Oliver Mortusewics, Lars Brautigam, Juha Rantala, Thomas Helleday. OXC-101 kills cancer by disturbing microtubule polymerization and inhibiting MTH1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1569.

Drug resistance and disease recurrence represent a major obstacle in the treatment of high-grade serous ovarian cancer (HGSOC). Here, we performed a comprehensive characterization, at the single-cell level, of residual (persister) cancer cells that survive neoadjuvant treatment in HGSOC and can give rise to future recurrences. We aim to understand the adaptive responses that enable persistence and the key pathways that underlie them to find new targets for combination therapy with the ultimate goal of eradicating all tumor cells. We collected 27 tumor samples (16 pre-treatment and 11 post-neoadjuvant carboplatin/taxol samples, with multi-site sampling per patient) for single-cell RNA sequencing (scRNA-Seq). To complement the patient-derived dataset and model the time course of drug persistence, we performed scRNA-Seq of 13 distinct ovarian cancer cell lines treated with chemotherapy in vitro for a series of time points up to 28 days. We performed single-cell gene expression and copy number analyses of patient samples, annotated detailed cell types, and detected multiple immune cell populations, the majority of which maintained a highly immunosuppressive program both in untreated and treated samples. Remarkably, we detected a rare population of cancer cells that differed dramatically in their transcriptional program. These cells exhibited high expression of components of motile cilia and the transcription factor FOXJ1, a master regulator of ciliogenesis. We validated the presence of FOXJ1+ cells in patient samples by immunohistochemistry and immunofluorescence. Ciliated cells were enriched in the post-treatment vs. pre-treatment samples, suggesting a possible role in drug persistence. We are now experimentally testing the role of the FOXJ1-driven transcriptional program in mediating drug resistance. To systematically identify transcriptional programs activated in persister cells, we performed differential expression analysis on pre- vs. post-treatment samples. We identified previously implicated mechanisms of drug resistance, such as the Epithelial-Mesenchymal Transition and the antiapoptotic response driven by MCL1. We additionally detected upregulation of the metallothionein family of genes, which may represent a mechanism of resistance to platinum-based compounds. We detected potentially novel mechanisms representing a stress response signature driven by JUND, an interferon gamma signature, and a pro-stem cell signature characterized by LGR5 expression. Independently, our analysis of the ovarian cancer cell lines treated with taxol in vitro confirmed the JUND transcriptional program as potentially driving ovarian cancer persistence. Our study will help reveal pathways enabling ovarian cancer cells to persist through treatment and, ultimately, could lead to novel approaches for targeting persistence and achieving deeper therapeutic responses. Citation Format: Elizaveta Leshchiner, Richard Panayiotou, Anay Gupta, Thomas Zhang, Kayli Neil, Nomeda Girnius, Aylin Henstridge, Brian P. Danysh, Ignaty Leshchiner, Sarah J. Hill, Laxmi Parida, Ursula A. Matulonis, Joan S. Brugge, Gad Getz. Drug persistence pathways in ovarian cancer identified by single-cell analysis of patient samples and cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3459.

Detection of multiple primary lung cancers is increasing in frequency, with up to 15% of all non-small cell lung cancer (NSCLC) patients presenting with two or more anatomically separate tumor nodules on CT scans. Increased detection is in part due to expanded lung cancer screening criteria in an aging population. Distinguishing multiple primary tumors from intrapulmonary metastases can be challenging, yet is critical for determining clinical management. Current models predict development of multiple primary tumors out of a cancerized field, such as occurs due to smoking. The occurrence of multiple primary tumors is unexplained in patients with EGFR-mutant lung cancer (~15% of all NSCLC), lacking environmental exposures. We identified patients with non-small cell lung cancer (NSCLC) who presented with multiple primary EGFR-mutant tumors, in the absence of family history of lung cancer or heavy smoking. We subjected the macrodissected tumors and surrounding normal tissues to whole exome sequencing as well as to analysis of hypermutable poly-guanine (poly-G) repeats, which are two orthogonal methods of lineage tracing. An additional familial case with a germline EGFR-T790M mutation was used to establish parameters for timing of somatic mutations, and functional properties of novel germline variants were modeled in vitro. Of eleven non-familial NSCLC cases with two or more geographically distinct EGFR-mutant lung cancers, two patients harbored a germline EGFR variant allele, which confers moderately enhanced signaling in vitro, followed by a somatically acquired EGFR mutation. In an additional four cases, both whole exome sequencing and poly-G repeat analyses indicate a distant shared somatic cell-of-origin, consistent with embryonic mosaicism. Three cases revealed clinically unappreciated metastasis and two cases remain unexplained. Together, our data suggest that multiple primary lung cancers with somatic EGFR driver mutations may result from genetic susceptibility, attributable either to attenuated EGFR germline variants or to embryonic mosaicism resulting in geographically disparate patches of predisposed lung tissue. Such predisposed cases should be surveilled for early detection of future tumors, and surgical resection in these cases should consider the life-long risk of additional cancers. Citation Format: Risa Burr, Ignaty Leshchiner, Christina L. Costantino, Martin Blohmer, Tilak Sundaresan, Justin Cha, Karsen Seeger, Sara Guay, Brian P. Danysh, Ira Gore, Raquel A. Jacobs, Kara Slowik, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Jaimie L. Barth, Taronish Dubash, Brian Chirn, Laxmi Parida, Lecia V. Sequist, Mari Mino-Kenudson, Kamila Naxerova, Shyamala Maheswaran, Gad Getz, Daniel A. Haber. Mechanisms of genetic predisposition to multifocal lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 771.

Responders to checkpoint blockade in Non Small Cell Lung Cancer (NSCLC) often feature an inflamed microenvironment prior to therapy. However, the complete set of molecular drivers connecting this histologic observation to enhanced tumor clearance remain enigmatic. In updated analysis of the Stand Up 2 Cancer-Mark Foundation (SU2C-MARK) Cohort - a collection of 393 patients with whole exome and/or RNA sequencing along with matched checkpoint blockade response annotation - we identify a prominent predictive role for inducible components of the immunoproteasome, a non-canonical peptide processing complex upstream of antigen presentation. Notably, these subunits are enriched as predictors relative to interferon-inducible genes as well as proteasome components in general, and are consistently associated with objective response, progression-free survival and overall survival. Expression of Immunoproteasome subunits associates positively with TCR (but not BCR) burden, supporting a mechanistic model in which enhanced immunoproteasome processivity leads to superior T-cell recognition. Furthermore, although they are known to be targets of interferon gamma (IFNɣ), we demonstrate that their expression is better modeled via a combination of IFNɣ and tumor necrosis factor-α (TNFα) levels, suggesting they may act as integrators of multiple cytokine cascades. Given the fact that the immunoproteasome can alter both antigen quantity as well as quality (including peptide cleavage site preference), the enhanced expression of this complex in the setting of checkpoint blockade response may have important implications for modeling of antigen presentation. These data also suggest novel strategies to enhance immune checkpoint blockade. Citation Format: Vivek Naranbhai, Arvind Ravi, Matthew Hellmann, Monica Arniella, Mark Holton, Samuel Freeman, Chip Stewart, Ignaty Leshchiner, Jaegil Kim, Yo Akiyama, Aaron Griffin, Natalie Vokes, Mustafa Sakhi, Vashine Kamesan, Hira Rizvi, Biagio Ricciuti, Patrick Forde, Valsamo Anagnostou, Jonathan Riess, Don Gibbons, Nathan Pennell, Vamsidhar Velcheti, Subba Digumarthy, Mari Mino-Kenudson, Andrea Califano, John Heymach, Roy Herbst, Julie Brahmer, Kurt Schalper, Victor Velculescu, Brian Henick, Naiyer Rizvi, Pasi Janne, Mark Awad, Andrew Chow, Benjamin Greenbaum, Marta Luksza, Alice Shaw, Jedd Wolchok, Nir Hacohen, Gad Getz, Justin Gainor. Immunoproteasome expression and checkpoint blockade response in advanced non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3468.

Background: It is estimated that 12.7% of the US population had been prescribed an antidepressant in the past month and that women are twice as likely to be on an antidepressant than men. Therefore, it is especially important to understand how SSRIs interact with female hormones and influence risk and progression of female cancers, especially breast cancer, being the most common. Up to a third of women use selective serotonin reuptake inhibitors (SSRIs) after breast cancer diagnosis. Recent investigation has demonstrated serotonin receptor (5-HTR2B) expression in the breast and identified serotonin production in breast tumor cells as an indicator of poor prognosis. This paper presents a unique comparative assessment between the influence of prior SSRI use, continuous and after use relative to breast cancer diagnosis on overall mortality among breast cancer patients. This analysis is expected to provide a clearer understanding of the influence of SSRIs based on period of use relative to time of diagnosis on overall mortality. Methods: The present study includes a population-based sample of consecutively diagnosed breast cancer cases identified as part of the Breast Cancer in Northern Israel study. Patients were recruited and followed from January 1st, 2000 to July, 2019. Participants completed risk factor questionnaires regarding medical, reproductive, family and personal history of cancer, medication use and health habits. Additionally full prescription data was available through the Israeli national CLALIT medical database. An analysis of 5976 newly diagnosed women with breast cancer was performed. K-M survival analysis and time-dependent and time-independent COX proportional hazard models were performed to determine overall survival based on interval of SSRI use. Results: Use of SSRIs in the 5 years prior to breast cancer diagnosis was associated with a 66% increase in overall mortality (HRadj.=1.66; CI: 1.05-2.63). Use of SSRIs with use that initiated after breast cancer diagnosis was associated with an 81% increase in mortality (HRadj.=1.81; CI: 1.58-2.06). Use of SSRIs in the 5 years post-diagnosis was associated with a significant (P5 years). For 24 months of SSRI use after diagnosis, there was a 99% increase in mortality (HR=1.99; CI: 1.39-2.83). Conclusion: SSRIs used both prior to and after breast cancer diagnosis are associated with reduced overall survival in breast cancer patients. The effect of SSRIs on mortality persisted even after adjustment for tamoxifen and factors thought to mediate the relationship behind depression and increased mortality including increased age at diagnosis, comorbidities and stage at diagnosis pointing toward other mechanisms mediating the association between SSRIs and impaired survival in breast cancer patients. Additional research is needed to better understand who is susceptible to the adverse effects of SSRIs on breast cancer overall mortality. Treating depressive symptomatology is of high importance. The results presented here indicate that risks and benefits of SSRI use after breast cancer diagnosis should be weighed when initiating pharmachotherapy and additional research is needed to better understand why SSRIs are associated with worse outcomes in breast cancer patients. Citation Format: Avital Fischer, Hedy Rennert, Ofra Barnett, Gad Rennert. The impact of SSRI use on overall survival in breast cancer patients in northern Israel [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-51.

Introduction:Breast cancer is among the three most common malignancies worldwide, and a better understanding of the epidemiology of the cardiovascular disease in females with breast cancer is needed Methods:Females with active breast cancer diagnosis hospitalized from January 2010 to December2017 were identified in the Nationwide Readmissions Database. Outcomes of interest were the prevalence of acute myocardial infarction (AMI), non-ST-segment myocardial infarction (NSTEMI), cardiac complications, and in-hospital mortality.Results:1,424,102 females with breast cancer were identified. Of whom, 17,578 (1.23%) were hospitalized for AMI. AMI patients were older with a mean age of 72.51 (11.73) vs. 62.22 (14.29), p 20102011201220132014201520162017P-trendCoronary Artery Disease8.1%8.3%8.0%8.5%8.9%8.1%9.7%10.2% Citation Format: Mohamed M Gad, Anas M Saad. Acute myocardial infarction prevalence and trends in females with active breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-26.